Flow cytometry system and method

ABSTRACT

A flow cytometry system having a flow channel defined through the thickness of a substrate is disclosed. Fluid flowing through the flow channel is illuminated by a first plurality of surface waveguides that are arranged around the flow channel in a first plane, while a second plurality of surface waveguides arranged around the flow channel in a second plane receive light after it has interacted with the fluid. The illumination pattern provided to the fluid is controlled by controlling the phase of the light in the first plurality of surface waveguides. As a result, the fluid is illuminated with light that is uniform and has a low coefficient of variation, improving the ability to distinguish and quantify characteristics of the fluid, such as cell count, DNA content, and the like.

FIELD OF THE INVENTION

The present invention relates to biotechnology in general, and, moreparticularly, to flow cytometry.

BACKGROUND OF THE INVENTION

Flow cytometry is a technique in which a fluid-flow system organizescells within a stream of fluid such that the cells pass in single-filethrough a detection zone. As the cells pass through the detection zone,they are illuminated by laser light, which scatters from each cell in amanner that depends on its structure. Most modern flow cytometryapproaches also employ numerous fluorochrome-labeled antibodies thatselectively bind with specific cellular features, such ascell-associated molecules, proteins or ligands. When excited by light attheir respective excitation wavelengths, each fluorochrome emits acharacteristic fluorescence signal, indicating the presence of thatfluorochrome-specific feature. The scattered light and fluorescencesignals are detected and analyzed to classify and/or count the cellsaccording to a set of parameters of interest. In some cases, onceclassified, the cells are sorted into sub-populations based on theirparticular characteristics.

Flow cytometry enables simultaneous multi-parameter analysis ofindividual cells in a fluid stream, such as analysis of cell surfacesand intracellular molecules, characterization and definition ofdifferent cell types in mixed cell populations, assessing the purity ofisolated subpopulations, and analyzing cell size and volume. Flowcytometers are used in many clinical and biological applications, suchas the diagnosis of blood cancers, basic research, clinical practice,and clinical trials.

Historically, fluid-flow systems in conventional flow cytometers havebeen of a stream-in-air configuration, in which the fluid stream isforced through a nozzle system so the cells pass in single file througha detection zone in open air. Other prior-art flow cytometers employ aflow cell configuration, wherein a sheath fluid hydrodynamically focusesthe sample fluid into the core of an open stream that traverses thedetection zone. Unfortunately, in each case, such prior-art flowcytometers have some significant disadvantages: (1) they are quiteexpensive; (2) they have a large footprint; (3) they are not easilyportable; and (4) they require extensive time, expertise, and expense touse and maintain. In addition, systems having an open-flow design aredifficult to adapt for use with infectious disease or pathogenicmicrobiological samples because of the risk of exposure.

To mitigate some of these disadvantages, microfluidics-based flowcytometers have been developed in which the sample fluid passes throughthe detection zone in an enclosed flow channel. The adoption ofmicrofluidics approaches also enables increased on-chip functionality,such as filtering, cell sorting, and overall flow control.

Microfluidics-based flow cytometers are disclosed, for example, in U.S.Patent Publication No. 2009/0051912, which describes a flow cytometersystem that is smaller and more portable than an open-flow system. Inoperation, the fluid-flow system is held under a microscope objective,which functions as an external optics system that provides the lightused to interrogate the cells and collect light scattered or emittedfrom the detection zone.

In fact, most conventional flow cytometers rely on external optics forilluminating the detection zone and/or detecting the scattered lightsignals. Unfortunately, this limits how small and portable a flowcytometer can be made. In addition, careful alignment between thefluid-flow system and the external optics is critical for realizingprecise and accurate measurements, and this alignment must be maintainedduring use to ensure proper system operation. Further exacerbating theseissues, the use of several fluorochromes usually gives rise to a needfor multiple lasers at different excitation wavelengths to excite thepallet of fluorochromes employed. Still further, numerouswavelength-filtered detectors are required to effectively discriminatebetween the resultant fluorescence signals. As a result, the use ofexternal optics can add significant cost to a flow cytometry system.

Integrating optical surface waveguides with microfluidics fluid-flowsystems offers some promise for mitigating some of the disadvantages ofexternal optics-based flow cytometers. Examples of a microfluidics-basedsystem having integrated optical surface waveguides are disclosed inU.S. Pat. No. 7,764,374, in which both fluid-flow channels andSU-8-based optical surface waveguides are formed on the top surface of asubstrate. One SU-8 surface waveguide emits light into an analysis zoneof the fluid-flow channel, while a second SU-8 surface waveguide,located across the fluid-flow channel, collects light after it haspassed through the analysis zone.

In similar fashion, U.S. Patent Publication No. 2013/0083315 disclosesflow cytometer arrangements having a first flow channel that includes adetection zone, and a plurality of “surface waveguide channels” that areadjacent to the detection zone. The surface waveguide channels arefilled with fluid that laterally guides light captured from thedetection zone to other regions of the substrate.

Unfortunately, such prior-art systems suffer from several disadvantages.It is often necessary to couple several independent light signals intoor out of a single region. SU-8-based surface waveguides andfluid-filled surface waveguides require significant chip real estate,however. As a result, forming more than few optical surface waveguidesthat access the same location can be challenging.

Further, flow cytometry performance is improved when the detection zoneis illuminated with substantially uniform light. Prior-art,microfluidics-based flow cytometers, however, are limited to providingillumination from one side of the fluid channel. As a result, uniformillumination of the sample fluid is precluded and system sensitivity isdegraded.

SUMMARY OF THE INVENTION

The present invention enables lab-on-a-chip systems having improvedillumination of a fluid stream and/or improved detection of lightsignals that arise from the fluid stream. As a result, embodiments ofthe present invention are able to provide better system performance,less measurement variation, and higher sensitivity than prior-artlab-on-a-chip systems. For example, lab-on-a-chip-based flow cytometersin accordance with the present invention can distinguish differentsubsets of cells with improved precision and can better quantifymeasurement parameters than flow cytometers known in the prior art.Although the present invention is particularly well suited for use inflow cytometers, it provides advantages in other lab-on-a-chip systemsas well, such as spectrometers, and the like.

An illustrative embodiment of the present invention is a flow cytometrysystem having a fluid channel formed through the thickness of asubstrate, and two sets of surface waveguides disposed on a surface ofthe substrate. Each set of surface waveguides is arranged such that itsend facets form a circular arrangement around the flow channel. Each setof surface waveguides is formed in a different plane that issubstantially orthogonal with the direction of fluid flow through thechannel.

A first set of surface waveguides is used to illuminate the detectionzone. Light from these excitation waveguides forms a substantiallyuniform illumination pattern in the flow channel. In some embodiments,the phase of the light in one or more of the excitation waveguides iscontrolled, thereby enabling control over the shape of the illuminationpattern in the detection zone.

The second set of surface waveguides is used to capture light after ithas interacted with the fluid in the detection zone. In someembodiments, the phase of the light in one or more of these collectionwaveguides is controllable. In some embodiments, at least one of thecollection waveguides is optically coupled with a wavelength filter thatdiscriminates spectral information in the light coupled into thatcollection waveguide.

In some embodiments, at least one set of surface waveguides is arrangedsuch that their facets form a polygonal arrangement around the flowchannel. In some of these embodiments, each side of the polygon includesa plurality of surface waveguide facets.

An embodiment of the present invention is an apparatus comprising: asubstrate that defines a first plane, the substrate comprising a flowchannel that is operative for conveying fluid along a first directionthat is substantially orthogonal to the first plane, the flow channelbeing located within a first region of the substrate; a first surfacewaveguide that is optically coupled with the flow channel, the firstsurface waveguide being located in a second plane within the firstregion, wherein the second plane is substantially parallel with thefirst plane; and a second surface waveguide that is optically coupledwith the flow channel in the first region, the second surface waveguidebeing located in a third plane within the first region, wherein thethird plane is substantially parallel with the second plane.

Another embodiment of the present invention is an apparatus comprising:a substrate having a thickness between a first major surface and asecond major surface; a first flow channel that is operative forconveying fluid through the thickness; a first plurality of surfacewaveguides, each of the first plurality of surface waveguides beingoptically coupled with the flow channel in a first region, the firstplurality of surface waveguides being coplanar in a first plane withinthe first region; and a second plurality of surface waveguides, each ofthe second plurality of surface waveguides being optically coupled withthe flow channel, the second plurality of surface waveguides beingcoplanar in a second plane within the first region; wherein, the firstmajor surface, the second major surface, the first plane, and the secondplane are substantially parallel.

Yet another embodiment of the present invention is a method comprising:conveying a first fluid along a first direction through a first region;interrogating the first fluid with a first illumination pattern that isbased on a first light signal emitted from a first surface waveguidethat lies in a first plane that is orthogonal to the first direction inthe first region; and coupling a first portion of the first illuminationpattern into a second surface waveguide that lies in a second plane thatis orthogonal to the first direction in the first region, wherein thefirst portion is coupled into the second surface waveguide after thefirst illumination pattern has interacted with the first fluid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C depict examples of prior-art microfluidic systems withintegrated surface waveguides.

FIG. 2 depicts a block diagram of a flow cytometer in accordance with anillustrative embodiment of the present invention.

FIG. 3 depicts operations of a method for performing flow cytometry inaccordance with the Illustrative embodiment.

FIG. 4 depicts a schematic drawing of a cross-sectional view of anoptofluidic system in accordance with the Illustrative embodiment of thepresent invention.

FIG. 5 depicts operations of a method for forming optofluidic system204.

FIG. 6 depicts a top view of optics plate 404.

FIGS. 7A-B depict top and cross-sectional views of region 602 of opticsplate 404.

FIG. 8 depicts sub-operations suitable for use in forming optics plate404.

FIG. 9A depicts a schematic drawing of a top view of region 602 afterthe definition of waveguide cores 706.

FIG. 9B depicts a schematic drawing of a top view of region 602 afterthe definition of waveguide cores 714.

FIG. 10 depict a side view of detection zone 414 during interrogation ofa cell 226.

FIG. 11 depicts a schematic drawing of a top view of region 602 inaccordance with a first alternative embodiment of the present invention.

FIGS. 12A-C depict simulated illumination patterns across detection zone1106 for different wavelengths of light.

FIGS. 13A-C depict plots of random phase field distribution acrossdetection zone 1106 for different wavelengths of excitation light.

FIG. 14 depicts an optics plate in accordance with a second alternativeembodiment of the present invention.

FIGS. 15A and 15B depict cross-section views of phase-control elements1402-E-i and 1402-C-i, respectively, in accordance with the secondalternative embodiment of the present invention.

FIG. 16 depicts simulation results for the change in effectiverefractive index for excitation waveguide core 706-i as a function ofthickness, length, and width of piezoelectric layer 1506.

FIG. 17 depicts a picture of a conventional flow cytometer flow cell inaccordance with the prior art.

FIGS. 18A-B depict a flow cytometry flow cell in accordance with a thirdalternative embodiment of the present invention.

DETAILED DESCRIPTION

FIGS. 1A-C depict examples of prior-art microfluidic systems withintegrated surface waveguides.

FIG. 1A depicts a portion of a Micro Total Analysis System (μTAS) thatincludes flow channel 102, illumination waveguide 104, and collectionwaveguide 106, all of which are formed on the top surface of substrate108. System 100 is in accordance with lab-on-a-chip (LOC) systemsdisclosed by J. Hubner, et al., in U.S. Pat. No. 7,764,374, issued Jul.27, 2010, which is incorporated herein by reference.

System 100 is an example of an absorption spectroscopy system. Inoperation, illumination waveguide 104 emits light signal 120 intoanalysis zone 118, which is defined by the area between illuminationwaveguide 104 and collection waveguide 106. Fluid 124 absorbs certaincharacteristic wavelengths of the light based on the constituents of thefluid. Some of the light not absorbed by the fluid is captured as lightsignal 122 by collection waveguide 106, which carries the light to awavelength dispersion system (not shown) that enables its spectralanalysis.

Illumination waveguide 104 and collection waveguide 106, as well as flowchannel 102, are formed on the top surface of substrate 108. Substrate108 typically comprises silicon, on which a layer of silicon dioxide(i.e., lower cladding 110) is formed as a lower cladding layer for thewaveguides. Substrate 108 defines substrate plane 114, which is alignedwith the x-y plane.

Each of the waveguides comprises a core region of SU-8 that issurrounded by layer 112, which acts to laterally confine light signals120 and 122 in the waveguides. Layer 112 is typically a layer of silicondioxide disposed on lower cladding layer 110. It should be noted thatlayer 112 defines plane 116, which is parallel to substrate plane 114.Flow channel 102 and waveguides 104 and 106 are all coplanar in plane116.

To complete the waveguide structures and enclose flow channel 102, asecond substrate (not shown for clarity) having a third layer of silicondioxide is bonded to layer 112.

While mitigating some of the drawbacks related to microfluidics-basedanalytical systems discussed above, system 100 still has somesignificant drawbacks. For example, by forming all surface waveguidesand flow channels such that they are coplanar, optical access toanalysis zone 118 is limited to primarily only one surface waveguidepair. As a result, simultaneous interrogation of analysis zone by morethan one light signal travelling along diverse paths is precluded. Inorder to interrogate fluid 110 with multiple light signals, therefore,multiple pairs of Illumination and collection waveguides are required,which leads to increased chip real estate for system 100 andcommensurately higher cost. Further, in applications where it isdesirable to collect light scattered by material in analysis zone 118,only forward-scattered light can be collected via a collectionwaveguide. Surface waveguides formed at positions to captureside-scattered light would, in general, be separated by a relativelylarge distance, making it difficult for a surface waveguide to capturesufficient light for a reliable measurement. Further, the use of SU-8 insystem 100 can lead to degradation over time, particularly when thesystem is used for short wavelengths and/or high intensities, due toabsorption of the light.

FIGS. 1B-C depicts schematic drawings of a top and cross-section view,respectively, of another example of a lab-on-a-chip system havingintegrated microfluidics and surface waveguides. System 126 is anexample of a portion of a partially integrated flow cytometer. System126 includes flow channel 128, surface waveguides 130-1 through 130-N,and lasers 132-1 through 132-N. System 126 is in accordance with flowcytometers described by C. Vannahme, et al., in “Plastic lab-on-a-chipfor fluorescence excitation with integrated organic semiconductorlasers,” Optics Express, Vol. 19, No. 9, pp. 8179-8186 (2011), which isincorporated herein by reference.

System 126 includes flow channel 128, surface waveguides 130 and laser132, all of which are monolithically integrated on substrate 138. Likesystem 100 described above, all flow channels and surface waveguides arecoplanar in substrate plane 150.

Substrate 138 is a poly(methyl methacrylate) (PMMA) substrate into whichflow channel 128 and depressions 140 are formed using conventionalplastic imprinting techniques. Depressions 140 are formed such that thebottom of each depression is characterized by a nascent gratingstructure 142, which is later coated with a thin film of organicsemiconductor tris(8-hydroxyquinoline)aluminum (Alq₃) to form organicsemiconductor lasers 132.

Surface waveguides 130 are formed directly in the PMMA material byexposing it to deep UV light, which breaks the molecular chains in thePMMA material to locally increase its refractive index. The unexposedPMMA retains its original, lower refractive index enabling it to serveas cladding material for the waveguides.

After the surface waveguides have been defined, PMMA cover 144 is joinedto substrate 138 to complete the fabrication of system 126.

In operation, lasers 132 are optically pumped to generate light signals146-1 through 146-N, which couple into surface waveguides 130-1 through130-N, respectively. Light signals 146 are used to excite the differentfluorochromes used to stain analytes in fluid 136. As the cells in thefluid flow sequentially through each of detection zones 134-1 through134-N, fluorochromes selectively bound to features of the cellsfluoresce at their characteristic fluorescence wavelengths as lightsignals 148-1 through 148-N.

Fluorescence signals 148 propagate out of plane 150 and are detected viaa free-space optics-based detection system.

The need to provide different detection zones so that multipleexcitation signals can be used to excite the full pallet offluorochromes adds significant complexity to system 126 and itsoperation. For example, because the fluorochromes are not excitedsimultaneously, ambiguity can creep into the measurement results.Further, the need for an external free-space detection system mitigatesmany of the benefits of Integrating flow channel 128 and surfacewaveguides 130. Still further, as discussed above, multiple detectionzones requires more chip real estate, which leads to higher system cost.

The present invention enables improved flow cytometry by arranging aplurality of surface waveguides in a plane that is not co-planar withthe direction in which a flow channel conveys a fluid. As a result, thefacets of the surface waveguides can be arranged on different sides ofthe flow channel. The present invention, therefore, enables greatercontrol over the manner in which the fluid is illuminated. It alsoimproves the ability to collect light from the flow channel by enablingcollection of light close to the flow channel even though the lightexits the flow channel along different directions.

It should be noted that, while the present invention is particularlywell suited for flow cytometry, it can also provide similar advantagesin other microfluidic applications, such as spectroscopy, chemicalsynthesis, capillary electrophoresis, lab-on-a chip applications, andthe like.

FIG. 2 depicts a block diagram of a flow cytometer in accordance with anillustrative embodiment of the present invention. Flow cytometer 200includes light source 202, optofluidic system 204, detector 206, andprocessor 208.

System 200 is operative for analyzing cells 226, which are contained inliquid-phase fluid 224. In some embodiments, system 200 is operative forother particles contained in a liquid-phase medium. In some embodiments,system 200 is operative for particles and/or cells contained in agas-phase medium (e.g., air, etc.). In some applications, system 200 isoperative for a gas-phase or liquid-phase fluids that are substantiallyparticle-free.

FIG. 3 depicts operations of a method for performing flow cytometry inaccordance with the illustrative embodiment. Method 300 begins withoperation 301, wherein optofluidic system 204 is provided. Method 300 isdescribed herein with continuing reference to FIG. 2, as well asreference to FIGS. 4-10.

Optofluidic system 204 is a monolithically integrated system thatincludes fluid-flow system 212 and detection system 214. Detectionsystem 214 comprises surface-waveguide-based excitation network 216,surface-waveguide-based collection network 218, and a portion offluid-flow system 212. Optofluidic system 204 is described in moredetail below and with respect to FIGS. 4-9.

FIG. 4 depicts a schematic drawing of a cross-sectional view of anoptofluidic system in accordance with the illustrative embodiment of thepresent invention. Optofluidic system 204 includes channel plates 402-1and 402-2, and optics plate 404. These plates collectively define eachof fluid-flow system 212 and detection system 214.

FIG. 5 depicts operations of a method for forming optofluidic system204. Method 500 begins with operation 501, wherein channel plates 402-1and 402-2 are formed.

Each of channel plates 402-1 and 402-2 is a conventional microfluidicchannel plate formed via conventional methods (e.g., reactive-ionetching (RIE), wet-chemical etching, sand-blasting, etc.). Channelplates 402-1 and 402-2 include channel networks 406-1 and 406-2,respectively, each of which is formed in a conventional planarprocessing substrate. Channel plates 402-1 and 402-2 also include vias410 and ports 412 distributed among the channel plates and optics plate404 to enable interconnection of the channel networks to each other andinterconnection of fluid-flow system 212 to external facilities, such asfluid sources, waste containers, etc.

Typically, for optical systems such as the illustrative embodiment, thechannel plate substrates are made of fused silica because it does notexhibit significant autofluorescence. In some applications, however, thechannel plate substrates comprise a material other than fused silica.Materials suitable for use in the channel plate substrates include,without limitation, glasses (e.g., silicon dioxide, borofloat glass,quartz, Pyrex, etc.), semiconductors (e.g., silicon, silicon carbide,germanium, GaAs, InP, etc.), metals, ceramics, plastics, compositematerials, and the like.

Each of channel networks 406-1 and 406-2 is a system of microfluidicchannels suitable for, in combination with flow channel 408, performingconventional fluidic operations on fluid 224, such as flow separation,filtering, mixing, sorting, etc., as well as forcing the cells in fluid224 to flow in single-file order through flow channel 408. The specificarrangement and functionality of channel networks 406-1 and 406-2 istypically a matter of application-based design. Channel networks 406-1and 406-2, flow channel 408, vias 410, and ports 412 collectively definefluid-flow system 212.

At operation 502, optics plate 404 is formed.

FIG. 6 depicts a top view of optics plate 404. Optics plate 404 includesflow channel 408, excitation network 216, collection network 218, flowchannel 408, and vias 410. In some embodiments, optics plate 404 is anelement provided as a portion of a conventional flow-cytometer flowchamber to, for example, improve or replace a conventional opticalexcitation or collection system.

Each of excitation network 216 and collection network 218 includes aplurality of N surface waveguides (referred to, individually, asexcitation waveguide 604-i or collection waveguide 606-i, where 1<i<N).Although N=8 in the illustrative embodiment, it will be clear to oneskilled in the art, after reading this Specification, how to specify,make, and use alternative embodiments of the present invention wherein Nis equal to any practical number, and can be as small as one. Further,one skilled in the art will recognize that excitation network 216 andcollection network 218 can include different numbers of surfacewaveguides.

In the illustrative embodiment, collection waveguides 606 are disposedin a plane located above excitation waveguides 604 in region 602. Insome embodiments, collection waveguides 606 are not disposed aboveexcitation waveguides. Further, in some embodiments, excitationwaveguides 604 lie in a plane that is above the plane of collectionwaveguides 606. Still further, in some embodiments, at least some ofexcitation waveguides 604 and collection waveguides 606 lie in the sameplane. In other embodiments, at least one of the pluralities ofexcitation waveguides 604 and collection waveguides 606 is distributedamong two or more waveguide layers.

FIGS. 7A-B depict top and cross-sectional views of region 602 of opticsplate 404. Region 602 provides a detailed view of flow channel 408,excitation waveguides 604-1 through 604-8, and collection waveguides606-1 through 606-8. Within region 602, excitation waveguides 604 andcollection waveguides 606 are formed on substrate 608 such that theirend facets are arranged in a substantially circular arrangement aboutthe center of flow channel 408. One skilled in the art will recognize,after reading this Specification, that the region of flow channel 408that is surrounded by the end facets of excitation waveguides 604 andcollection waveguides 606 substantially defines detection zone 414.

FIG. 8 depicts sub-operations suitable for use in forming optics plate404. Operation 502 begins with sub-operation 801, wherein core layer 702is formed on the top surface of substrate 608.

Substrate 608 is a planar substrate that is analogous to the channelplate substrates described above and, in the Illustrative embodiment,comprises fused silica in order to suppress autofluorescence. In someembodiments, however, substrate 608 can comprise another material, asdiscussed above and with respect to channel plates 402-1 and 402-2.Substrate 608 defines substrate plane 610, which lies generally in thex-y plane, as indicated.

Core layer 702 is a conventional planar layer of stoichiometric siliconnitride, deposited on the top surface of substrate 608 usinglow-pressure chemical vapor deposition (LPCVD). Core layer 702 has athickness of approximately 25 nanometers (nm) and defines waveguideplane 704. In some embodiments, core layer 702 has a differentthickness. In some embodiments, core layer 702 comprises a materialother than silicon nitride. Materials suitable for use in core layer 702include any material through which excitation signals can propagate. Insome embodiments, core layer 702 is formed with a different suitableformation process.

In some embodiments, substrate 608 includes a surface layer, such as asilicon oxide, that functions as a lower cladding layer for waveguidesformed from core layer 702.

At operation 802, core layer 702 is patterned in conventional fashion todefine waveguide cores 706-1 through 706-8 (referred to, collectively,as waveguide cores 706). Typically, core layer 702 is patterned viaphotolithography and RIE. Waveguide cores 706 are patterned such thateach has a width within the range of approximately 1 micron toapproximately 4 microns, and typically approximately 2 microns. As aresult, each of waveguide cores 706 defines a stripe waveguide that issuitable for single-mode operation at the wavelengths of light includedin excitation light 210. Each of waveguide cores 706 includes an endfacet 708, the plurality of which is arranged in a substantiallycircular arrangement about detection zone 414.

In some embodiments, at least one of waveguide cores 706 comprisesdifferent materials, is of a different waveguide type, and/or hasdifferent dimensions (i.e., thickness or width) than another of surfacewaveguide cores 706. Different core materials, types, and/or dimensionsenable surface waveguides that are preferable for, for example,different wavelengths, diverse functions (e.g., providing light to orcollecting light from detection zone 414, etc.), and the like. In someembodiments, therefore, at least one of operation 801 and 802 isrepeated one or more times.

FIG. 9A depicts a schematic drawing of a top view of region 602 afterthe definition of waveguide cores 706.

At operation 803, intermediate cladding 710 is formed in conventionalfashion. Intermediate cladding 710 is a layer of silicon dioxidedeposited via LPCVD. Typically, after formation, intermediate cladding710 is planarized via chemical-mechanical polishing, or another suitableplanarization technique. Intermediate cladding 710 has a thickness thatis typically within the range of approximately 1 microns toapproximately 30 microns. Intermediate cladding 710 operates as both anupper cladding for excitation waveguides 604 and a lower cladding fordetection waveguides 606.

One skilled in the art will recognize that the thickness of intermediatecladding 710 is a matter of design and is based on several factors, suchas the acceptable level of cross-talk between excitation network 216 andcollection network 218, acceptable levels of loss in the excitation andcollection waveguides, and the like.

In some embodiments, intermediate cladding 710 is formed via anotherdeposition technique, such as plasma-enhanced chemical vapor deposition(PECVD), sputtering, spin-on glass deposition, and the like. In someembodiments, intermediate cladding 710 comprises a material other thansilicon dioxide. One skilled in the art will recognize that the choiceof material for intermediate cladding 710 is based on numerous factors,including the wavelength of light, the materials of substrate 608 andcore layers 702 and 712, material compatibility with fluid 224, etc.

At operation 804, core layer 712 is formed on Intermediate cladding 710.Core layer 712 is analogous to core layer 702 described above; however,core layer 712 is formed such that it has a thickness of approximately100 nm. Core layer 712 defines waveguide plane 716.

At operation 805, core layer 712 is patterned in conventional fashion todefine stripe waveguide cores 714-1 through 714-8 (referred to,collectively, as waveguide cores 714), which collectively definewaveguide plane 716. Waveguide plane 716 is substantially parallel withsubstrate plane 610. Typically, core layer 712 is patterned viaphotolithography and RIE.

In order to facilitate collection of light from detection zone 414,waveguide cores 714 are patterned such that they have width within therange of approximately 1 micron to approximately 4 microns, andtypically approximately 2 microns. As a result, each of waveguide cores714 operates as a multimode waveguide core for the wavelengths of lightin collected light 220. Like waveguide cores 706, waveguide cores 714have end facets 718, which are arranged in a substantially circulararrangement about detection zone 414. In some embodiments, waveguidecores 714 have a different width or height.

The dimensions for waveguide cores 706 (and/or waveguide cores 714provided herein are merely exemplary. One skilled in the art willrecognize that the specific dimensions of a waveguide depend on systemand application considerations, and that any suitable dimensions forthese waveguides is within the scope of the present invention.

Further, as discussed above vis-à-vis waveguide cores 706, in someembodiments, at least one of waveguide cores 714 comprises differentmaterials, is of a different waveguide type, and/or has differentdimensions (i.e., thickness or width) than another of surface waveguidecores 714. Different core materials, types, and/or dimensions enablesurface waveguides that are preferable for, for example, differentwavelengths, diverse functions (e.g., providing light to or collectinglight from detection zone 414, etc.), and the like. In some embodiments,therefore, at least one of operation 804 and 805 is repeated one or moretimes.

FIG. 9B depicts a schematic drawing of a top view of region 602 afterthe definition of waveguide cores 714.

At operation 806, top cladding layer 720 is formed on waveguide cores714 to complete formation of collection waveguides 606. Top claddinglayer 720 is analogous to intermediate cladding layer 710.

It should be noted that, at the end of operation 806, each of waveguidecores 706 and 714 extends past perimeter 902, which defines the extentflow channel 408 as it will be formed in operation 807. This ensuresthat each of the pluralities of end facets 708 and 718 will be arrangedin a circular pattern located at the edge of the flow channel once it isformed, since the end facets are formed by the deep-RIE process used toform the flow channel. In some embodiments, the end facet of at leastone surface waveguide is not formed during the operation in which flowchannel 408 is formed. In some embodiments, one or more of end facets708 and 718 is formed when its respective core layer is patterned todefine its corresponding waveguide cores.

Although the Illustrative embodiment comprises excitation waveguidesthat operate as single-mode waveguides and collection waveguides thatoperate as multimode waveguides, it will be clear to one skilled in theart, after reading this Specification, how to specify, make, and usealternative embodiments of the present invention comprising at least oneexcitation waveguide that operates as a multimode waveguide and/or atleast one collection waveguide that operates as a single-mode waveguide.

In some embodiments, collection waveguides 606 are routed individuallyto the edge of substrate 608 and detected independently. Such anarrangement can, for example, enable maintenance of angle-dependentscattering information.

At operation 807, flow channel 408 is formed through the thickness ofsubstrate 608 and its surface layers. Flow channel 408 has a diameterwithin the range of approximately 20 microns to approximately 120microns, and is typically approximately 40 microns, which restrictscells in fluid 224 to single-file flow through detection zone 414. Itshould be noted that the present invention is applicable to applicationsother than flow cytometry, wherein the size of flow channel 408 does notnecessarily restrict the size of particles or cells in fluid 224. Oneskilled in the art will recognize, therefore, that the diameter of flowchannel 408 is a matter of application-based design considerations.

Although in the illustrative embodiment, flow channel 408 is formed viaconventional deep-RIE, it will be clear to one skilled in the art, afterreading this Specification, how to specify, make, and use alternativeembodiments wherein flow channel 408 is formed via a different process,such as sand blasting, laser ablation, wet etching, etc., are alsosuitable for the formation of the flow channel depending on thematerials used in system 200.

Returning now to method 500, at operation 503, channel plates 402-1 and402-2 are joined with optics plate 404 such that channel networks 406-1and 406-2 are fluidically coupled with flow channel 408. In theillustrative embodiment, the plates are joined using a wafer bondingtechnique, such as fusion bonding, thermo-anodic bonding, etc., to fusethe three plates into a single monolithic element. In some embodiments,the plates are joined via another suitable method, such as clamping,etc. In some embodiments, a fluidic seal is formed between the fluidicelements on each plate using intervening elements, such as O-rings,gaskets, deposited material (e.g., polyimide, SU-8, PMMA, etc.) and thelike.

At operation 504, ports 414 are fluidically coupled with externalfluidic systems, such as a reservoir, pumping system, and wastecontainer for fluid 224.

At operation 505, excitation network is optically coupled with lightsource 202.

Light source 202 is a conventional multi-spectral light source thatprovides excitation light having wavelengths suitable for exciting thefull pallet of fluorochromes used during operation of flow cytometer200. In some embodiments, light source 202 includes a plurality of lightemitting devices and/or spectral filters, such as lasers, light-emittingdiodes (LEDs), superluminescent diodes, and the like.

At operation 506, collection network is optically coupled with detector206.

Detector 206 is a conventional detection system operative for detectingone or more of the wavelengths included in collected light 220, which isreceived from optofluidic system 204. Detector 206 includes a pluralityof detectors and wavelength filters suitable for discriminatingfluorescence signals and scattered signals collected by collectionnetwork 216. Detector 206 provides output signal 222 to processor 208.

Processor 208 is a conventional processing system operative forreceiving output signal 222 and conducting analysis of the output signalto estimate one or more parameters of fluid 224 and/or cells 226.

Returning now to method 300, at operation 302, fluid 224 is pumpedthrough optofluidic system 212, from reservoir 228 to waste container230, such that its constituent cells 226 flow through detection zone 414along flow direction 724. Flow direction 724 is aligned with thez-direction, as depicted in FIG. 7B, which is orthogonal to each ofsubstrate plane 610, and waveguide planes 704 and 716. In someembodiments, flow direction 724 is not orthogonal with waveguide planes704 and 716; however, it should be noted that it is an aspect of thepresent invention that flow direction 724 is neither parallel norcoplanar with either of the waveguide planes.

At operation 303, light source 202 provides light signal 210 tooptofluidic system 204.

At operation 304, cells 226 are interrogated with excitation light 210.

FIG. 10 depicts a side view of detection zone 414 during interrogationof a cell 226. Excitation light is provided to cell 226 by excitationwaveguides 604.

Interrogation of cell 226 with excitation light 210 gives rise to outputlight 1002, which includes forward-scattered, side-scattered, andfluorescent light signals as discussed above.

At operation 305, collection waveguides 606 capture a portion of outputlight 1002 as collected light 220.

At operation 306, collection waveguides 606 convey collected light 220to detector 206, which converts it into output signal 222.

At operation 307, processor 208 performs analysis of output signal 222and provides an estimate of the parameters of interest for cells 226.

FIG. 11 depicts a schematic drawing of a top view of region 602 inaccordance with a first alternative embodiment of the present invention.System 1100 is analogous to system 200 described above; however, system1100 includes excitation and collection waveguide pairs that arearranged in arrays that collectively form a polygonal arrangement thatsurrounds flow channel 408.

Each waveguide pair 1102 includes one excitation waveguide 604 and onecollection waveguide 606, as described above and with respect to FIGS.7A-B.

Waveguide pairs 1102 are arranged in waveguide arrays 1104-1 through1104-8 (referred to, collectively, as waveguide arrays 1104), each ofwhich includes eight waveguide pairs arranged in linear fashion.Waveguide arrays 1104 form an octagonal arrangement that is concentricwith flow channel 408. Although in the example shown, waveguide arrays1104 form a polygon having eight sides, it will be clear to one skilledin the art, after reading this Specification, how to specify, make, anduse alternative embodiments wherein waveguide arrays 1104 form a polygonhaving any practical number of sides. Further one skilled in the artwill recognize that waveguide arrays 1104 can include any practicalnumber of waveguide pairs.

The arrangement of waveguide arrays 1104 about flow channel 408 givesrise to detection zone 1106 having a substantially circularcross-section. In some embodiments, detection zone 1106 has across-sectional shape other than circular.

FIGS. 12A-C depict simulated illumination patterns across a diameter ofa detection zone for different wavelengths of excitation light.

Patterns 1200, 1202, and 1204 show the illumination pattern across a120-micron diameter flow channel for TM-polarized light at wavelengthsof 404, 532, and 632 nm, respectively.

FIGS. 13A-C depict plots of random phase field distribution acrossdetection zone 1106 for different wavelengths of excitation light.

Plots 1300, 1302, and 1304 depict the distribution of optical poweracross a 160-micron diameter flow channel for TE-polarized light atwavelengths of 404, 532, and 632 nm, respectively.

FIGS. 12 and 13 evince that substantially uniform illumination can berealized by providing excitation light from an octagonal pattern ofwaveguide arrays in accordance with the first illustrative embodiment.

Although the waveguide arrangements described above enable significantimprovement in Illumination of a flow channel region, the illuminationpattern for any wavelength is determined purely by the arrangement ofthe facets about the region and are not controllable during operation.It would be desirable to enable control over the shape of theillumination pattern during use, however.

It is another aspect of the present invention that control over theillumination pattern in the detection zone can be gained by controllingthe phase and/or amplitude of the light launched by one or moreexcitation waveguides into detection zone 414. In some embodiments, thisenables beam shaping capable of providing specific illumination patternshaving local intensity maxima at discrete positions within detectionzone 414.

Further, identification of the light signals captured by an Individualcollection waveguide can also be improved by controlling the phase ofthe light signal in that waveguide.

In some embodiments, at least one of collection waveguides 606 is asingle-mode waveguide that includes a polarization filter. Further, insome of these embodiments, excitation light 210 is provided is polarized(e.g., as TM light). In such a configuration, the present inventionenables detection of light that is partially converted to anotherpolarization mode (e.g., TE), which provides an Indication as toparticle shape (e.g., ratio of diameter versus length, etc.), asdescribed by N. G. Khlebtsov, et al., in “Can the Light Scatteringdepolarization Ratio of Small Particles Be Greater Than ⅓?” J. Phys.Chem. B, Vol. 2005, No. 109, pp. 13578-13584 (2005), which isincorporated herein by reference.

FIG. 14 depicts an optics plate in accordance with a second alternativeembodiment of the present invention. Optics plate 1400 is analogous tooptics plate 404 and includes all of the same structure; however, opticsplate 1400 also includes phase-control elements 1402-E-1 through1402-E-8 and phase-control elements 1402-C-1 through 1402-C-8 (referredto, collectively, as PCE 1402-E and PCE 1402-C, respectively). Each ofphase-control elements 1402-E and 1402-C is operatively coupled with anexcitation waveguide or collection waveguide such that it can controlthe phase of a light signal propagating through the waveguide.

FIGS. 15A and 15B depict cross-section views of phase-control elements1402-E-i and 1402-C-i, respectively, in accordance with the secondalternative embodiment of the present invention. Each of phase-controlelements 1402-E-i and 1402-C-i comprises strain element 1502 that isoperatively coupled with its respective waveguide core. Strain element1502 includes lower electrode 1504, piezoelectric layer 1506, and upperelectrode 1508.

Each of lower electrode 1504 and upper electrode 1508 is a layer ofelectrically conductive material, such as platinum, gold, aluminum, etc.The thickness of lower electrode 1504 and upper electrode 1508 is amatter of design choice.

Piezoelectric layer 1506 is a layer of piezoelectric material, such aslead zirconate titanate (PZT), having thickness, t. Piezoelectric layer1506 is patterned to form a substantially rectangular region on whichupper electrode 1508 is formed. One skilled in the art will recognizethat the width, w, and length, L, of upper electrode 1508 (where w isthe dimension of the layer along the direction transverse to the axialdirection of Its underlying waveguide and L is the dimension of thelayer along the axial direction of its underlying waveguide) effectivelydefine the operative dimensions of strain element 1502. As discussedbelow and with respect to FIG. 16, the operational characteristics ofPCE 1402-E and PCE 1402-C are based on the values of t, w, and L.

In some embodiments, one or both of piezoelectric layer 1506 and lowerelectrode 1504 are not patterned and, therefore, remain over the entiresurface of the substrate. In such embodiments, vias are formed throughthe piezoelectric material to enable electrical contact to beestablished to the underlying lower electrode.

In PCE 1402-E-i, strain element 1502 is disposed on intermediatecladding 710 in a region where core layer 712 has been removed duringpatterning of collection waveguide cores 714.

In PCE 1402-C-i, strain element 1502 is disposed upper cladding 722 in aregion where core layer 702 has been removed during patterning ofexcitation waveguide cores 706.

Processor 208 provides control signals 1510-E-i and 1510-C-i to each ofPCE 1402-E-i and PCE 1402-C-i, respectively. These control signals applya voltage differential between lower electrode 1504 and upper electrode1508, which gives rise to strain in piezoelectric layer 1506. Thisstrain is transmitted into the underlying waveguide core, resulting in achange in its effective refractive index.

In similar fashion, the phase of light propagating through collectionwaveguide 606-1 is controlled by control signal 1508-C-i, provided byprocessor 208. Control signal 1508-C-i is a voltage differential appliedto lower electrode 1504 and upper electrode 1508, disposed on uppercladding 722, as shown. Application of a voltage differential to theelectrodes of PCE 1402-C-i give rise to strain in piezoelectric layer1506, which is transmitted into waveguide guide core 714-i, resulting ina change in its refractive index.

FIG. 16 depicts simulation results for the change in effectiverefractive index for excitation waveguide core 706-i as a function ofthe thickness of piezoelectric layer 1506 and the length, L, and width,w, of upper electrode 1508.

Plot 1600 shows the change in refractive index for excitation waveguidecore 706-i, as a function of upper electrode width, w, for apiezoelectric layer 1506 having a thickness of 0.5 micron and a upperelectrode length of 13.99 mm.

Plot 1602 shows the change in refractive index for excitation waveguidecore 706-i, as a function of upper electrode width, w, for apiezoelectric layer 1506 having a thickness of 1.0 micron and a upperelectrode length of 7.66 mm.

Plot 1604 shows the change in refractive index for excitation waveguidecore 706-i, as a function of upper electrode width, w, for apiezoelectric layer 1506 having a thickness of 1.5 micron and a upperelectrode length of 5.51 mm.

Plot 1606 shows the change in refractive index for excitation waveguidecore 706-i, as a function of upper electrode width, w, for apiezoelectric layer 1506 having a thickness of 2.0 micron and a upperelectrode length of 4.45 mm.

Plots 1600 through 1606 that a significant change in refractive indexcan be achieved in a waveguide core operatively coupled with strainelement 1502, which will give rise to a commensurate change in phase fora light signal propagating through the waveguide.

One skilled in the art will recognize that piezoelectric-layer-based PCE1042 is merely one example of a phase control element within the scopeof the present invention. For example, phase can be controlled viathermo-optic modulation (I.e., via a heater disposed on a waveguide),birefringence modulation using a magnetostrictive element, etc. Further,in some embodiments, control over the illumination pattern in thedetection zone is provided by controlling amplitude of the lightlaunched by one or more excitation waveguides using an amplitudemodulator, such as a Mach-Zehnder interferometer structure.

In some embodiments, at least some of excitation waveguides 604 includephase and/or amplitude controllers such that the excitation waveguidesare operative for steering an illumination pattern around detection zone414. As a result, a single fixed-location collection waveguide can beused to collect scattered/fluorescent light from the detection zone. Insome cases, this affords a simpler detection scheme and/or enables theuse of a single large and sensitive detector (e.g., an avalanchephotodiode, photomultiplier tube, etc.) APD to detect output opticalsignals that are too weak to collect with a conventional detector array.By correlating the detected light with the direction of the steeredillumination pattern, angular information is retained.

FIG. 17 depicts a picture of a conventional flow cytometer flow cell inaccordance with the prior art. Flow cell 1700 includes cell body 1702,channel 1704, fluid port 1706, and lens 1708.

Channel 1704 is formed through the length of cell body 1702 such that itdefines a long conduit for conveying fluid through detection zone 414.Detection zone 414 is defined by the position of lens 1708, which isintegrated into the flow cell such that it focuses free-space excitationlight into the detection zone and collects light (e.g., forward- andside-scattered light and fluorescence signals) from the detection zone.

FIGS. 18A-B depict a flow cytometry flow cell in accordance with a thirdalternative embodiment of the present invention. Flow cell 1800represents embodiments of the present invention that have substantiallythe same form factor as prior-art flow cells, but afford improvedoptical system performance and simpler operation. Flow cell 1800comprises cell body 1802, channel 1804, fluid port 1706, and optofluidicsystem 1806.

Cell body 1802 is analogous to cell body 1702; however cell bodyincludes two conventional cell body portions 1702-1 and 1702-2, whichare attached to either side of optofluidic system 1806. Typically, cellbody portions 1702-1 and 1702-2 are joined with optofluidic system 1806via a conventional joining technology, such as fusion bonding, glue,etc. Cell body portions 1702-1 and 1702-2 include sections of channel1704, which bookend and fluidically couple flow channel 408 tocollectively define channel 1804.

Optofluidic system 1806 is analogous to optics plate 404 and comprisesplate 1808 and detection system 1812, which includes excitation network1814, collection network 1816, and flow channel 408. Excitation network1814 and collection network 1816 are analogous to excitation network 216and collection network 218 and are formed in waveguide plane 1810, whichis defined by the top surface of plate 1808.

It should be noted that the waveguides of excitation network 1814 andcollection network 1816 are formed in the same waveguide plane. As aresult, all of the excitation waveguides are arranged about one side offlow channel 408, while all of the collection waveguides are arrangedabout the other side of the flow channel. While the illumination patternprovided by such an arrangement is not as uniform as in some otherembodiments of the present invention, it still typically represents asignificant improvement over illumination patterns provided by prior-artflow cytometer arrangements (e.g., that shown of system 1700). In someembodiments, the waveguides of excitation network 1814 and collectionnetwork 1816 are disposed in two or more waveguide planes, as describedabove. Further, in some embodiments, the waveguides of excitationnetwork 1814 and collection network 1816 are arranged in anotherarrangement about flow channel 408, such as those arrangements describedabove.

One skilled in the art will recognize that there are several ways tooptically couple to and from excitation network 1814 and collectionnetwork 1816, such as butt coupling optical fibers to the waveguidenetworks, focusing free-space optical signals into the end facets of thenetworks, etc.

It is to be understood that the disclosure teaches just one example ofthe illustrative embodiment and that many variations of the inventioncan easily be devised by those skilled in the art after reading thisdisclosure and that the scope of the present invention is to bedetermined by the following claims.

What is claimed is:
 1. An apparatus comprising: a substrate that definesa first plane, the substrate comprising a flow channel that is operativefor conveying fluid along a first direction that is substantiallyorthogonal to the first plane, the flow channel being located within afirst region of the substrate; a first surface waveguide that isoptically coupled with the flow channel, the first surface waveguidebeing located in a second plane within the first region, wherein thesecond plane is substantially parallel with the first plane, wherein thefirst surface waveguide is operatively coupled with a first phasecontroller that is operative for controlling the phase of a first lightsignal propagating in the first surface waveguide; and a second surfacewaveguide that is optically coupled with the flow channel in the firstregion, the second surface waveguide being located in a third planewithin the first region, wherein the third plane is substantiallyparallel with the second plane.
 2. The apparatus of claim 1, wherein thesecond plane and the third plane are the same plane.
 3. The apparatus ofclaim 1, further comprising: a first plurality of surface waveguidesthat includes the first surface waveguide, each of the first pluralityof surface waveguides being located in the second plane in the firstregion and being optically coupled with the flow channel; and a secondplurality of surface waveguides that includes the second surfacewaveguide, each of the second plurality of surface waveguides beinglocated in the third plane in the first region and being opticallycoupled with the flow channel.
 4. The apparatus of claim 3, wherein thefirst plurality of surface waveguides is arranged in a circulararrangement about the flow channel in the first region.
 5. The apparatusof claim 4, wherein the second plurality of surface waveguides isarranged in a circular arrangement about the flow channel in the firstregion.
 6. The apparatus of claim 3, wherein the first plurality ofsurface waveguides is arranged in a polygonal arrangement about the flowchannel in the first region, the polygonal arrangement having aplurality of sides, each having at least one surface waveguide of thefirst plurality thereof.
 7. The apparatus of claim 6, wherein the secondplurality of surface waveguides is arranged in a polygonal arrangementabout the flow channel in the first region, the polygonal arrangementhaving a plurality of sides, each having at least one surface waveguideof the second plurality thereof.
 8. The apparatus of claim 1, whereinthe second surface waveguide is operatively coupled with a second phasecontroller that is operative for controlling the phase of a second lightsignal propagating in the second surface waveguide.
 9. The apparatus ofclaim 1, wherein the first phase controller comprises: a surfacewaveguide core comprising a first material, the surface waveguide corebeing optically coupled with the first surface waveguide; an uppercladding comprising a second material; a first electrode; a secondelectrode; and a first layer comprising a piezoelectric material, thefirst layer being between the first electrode and second electrode;wherein the first layer is operative for inducing a strain in thesurface waveguide core when a voltage is applied between the first andsecond electrodes.
 10. The apparatus of claim 9, wherein thepiezoelectric material comprises lead zirconium titanate.
 11. Theapparatus of claim 9, wherein the substrate comprises fused silica. 12.An apparatus comprising: a substrate having a thickness between a firstmajor surface and a second major surface; a first flow channel that isoperative for conveying fluid through the thickness; a first pluralityof surface waveguides, each of the first plurality of surface waveguidesbeing optically coupled with the flow channel in a first region, thefirst plurality of surface waveguides being coplanar in a first planewithin the first region; a first phase controller, the first phasecontroller being optically coupled with a first surface waveguide of thefirst plurality thereof, the first phase controller being operative forcontrolling the phase of a first light signal propagating in the firstsurface waveguide; and a second plurality of surface waveguides, each ofthe second plurality of surface waveguides being optically coupled withthe flow channel, the second plurality of surface waveguides beingcoplanar in a second plane within the first region; wherein, the firstmajor surface, the second major surface, the first plane, and the secondplane are substantially parallel.
 13. The apparatus of claim 12, whereinthe first flow channel is operative for conveying the fluid along afirst direction that is substantially orthogonal to the first majorsurface.
 14. The apparatus of claim 12, wherein the first plurality ofsurface waveguides is operative for generating an optical pattern in theflow channel.
 15. The apparatus of claim 14, wherein the first pluralityof surface waveguides is operative for controlling the shape of theoptical pattern.
 16. The apparatus of claim 12, wherein at least one ofthe second plurality of surface waveguides is operatively coupled with awavelength filter.
 17. The apparatus of claim 12, wherein the firstplurality of surface waveguides is arranged in a circular arrangementabout the flow channel in the first region.
 18. The apparatus of claim12, wherein the first plurality of surface waveguides is arranged in apolygonal arrangement about the flow channel in the first region, thepolygonal arrangement having a plurality of sides, each side having atleast one surface waveguide of the first plurality thereof.
 19. Theapparatus of claim 12, wherein the first phase controller comprises: asurface waveguide core comprising a first material, the surfacewaveguide core being optically coupled with the first surface waveguide;an upper cladding comprising a second material; a first electrode; asecond electrode; and a first layer comprising a piezoelectric material,the first layer being between the first electrode and second electrode;wherein the first layer is operative for inducing a strain in thesurface waveguide core when a voltage is applied between the first andsecond electrodes.
 20. The apparatus of claim 19, wherein thepiezoelectric material comprises lead zirconium titanate.
 21. Theapparatus of claim 19, wherein the substrate comprises fused silica. 22.A method comprising: conveying a first fluid along a first directionthrough a first region; interrogating the first fluid with a firstillumination pattern that is based on a first light signal emitted froma first surface waveguide that lies in a first plane that is orthogonalto the first direction in the first region; controlling the shape of thefirst illumination pattern by controlling the phase of the first lightsignal; and coupling a first portion of the first illumination patterninto a second surface waveguide that lies in a second plane that isorthogonal to the first direction in the first region, wherein the firstportion is coupled into the second surface waveguide after the firstillumination pattern has interacted with the first fluid.
 23. The methodof claim 22 further comprising determining a characteristic of the firstfluid based on the first portion.
 24. The method of claim 22 furthercomprising: providing a first plurality of surface waveguides thatincludes the first surface waveguide, wherein the first plurality ofsurface waveguides is provided such that it is substantially coplanar inthe first plane in the first region; and providing a first plurality oflight signals that includes the first light signal, wherein each of thefirst plurality of light signals is emitted by a different surfacewaveguide of the first plurality thereof; wherein the first illuminationpattern is based on the first plurality of light signals.
 25. The methodof claim 24 further comprising: providing a second plurality of surfacewaveguides that includes the second surface waveguide, wherein thesecond plurality of surface waveguides is provided such that it issubstantially coplanar in the second plane in the first region; andcoupling a second portion of the first illumination pattern into a thirdsurface waveguide that is included in the second plurality of surfacewaveguides, wherein the second portion is coupled into the third surfacewaveguide after the first illumination pattern has interacted with thefirst fluid.
 26. The method of claim 24 wherein the phase of the atleast one of the first plurality of light signals is controlled bycontrolling a strain applied to at least one of the first plurality ofsurface waveguides.
 27. The method of claim 26 wherein the strain isapplied to the at least one of the first plurality of surface waveguidesby providing a voltage differential across a first layer comprising apiezoelectric material, the first layer being operatively coupled withat least one of the first plurality of surface waveguides.